Hybridoma cell lines and monoclonal antibodies to theophylline

ABSTRACT

Monoclonal antibodies to theophylline having 5% or less cross-reactivity with caffeine and the continuous hybrid monoclonal cell lines for their production are provided. These antibodies are useful in a particle-enhanced turbidimetric inhibition immunoassay for theophylline.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of application Ser. No.393,680, filed June 30, 1982, now abandoned.

TECHNICAL FIELD

This invention relates to hybrid cell lines (lymphocyte hybridomas) forthe production of monoclonal antibody to theophylline(1,3-dimethylxanthine), to the homogeneous, monospecific antibodies, andtheir use in immunoassays for theophylline.

BACKGROUND ART

In 1975, Kohler and Milstein reported the establishment of a continuoushybrid cell line (hybridoma) derived by the fusion of murine myelomacells to spleen cells from an immunized mouse which secreted monoclonalantibody to sheep red blood cells; Nature, Volume 256, 495 (1975).Numerous publications have since appeared describing the production ofmonoclonal antibodies to other antigens and haptens. See, for example,Current Topics in Microbiology and Immunology, Volume 81, F. Melchers,M. Potter, and N. Warner, ed., Springer-Verlag, 1978, and referencescontained therein; and Monoclonal Antibodies, R. Kennett, T. McKearn andK. Bechtol, ed., Plenum Press, 1980, and references contained therein.

Although the general technique of producing hybridomas is well known andunderstood, there are still considerable difficulties involved inproducing and selecting a hybridoma cell line secreting antibody havinga given set of desired properties.

European Patent Application No. 25,722, published Mar. 25, 1981,discloses the production of a monoclonal antibody to a humanT-lymphocyte cell surface antigen.

The production of monoclonal antibodies to the cardiac glycoside,digoxin, has been reported in Federation Proceedings, Volume 39, 928(1980) and in Scand. J. clin. Lab. Invest., Volume 41, 75 (1981).

There is a rapidly expanding market for clinical diagnostic assays whichcan be used to monitor the levels of various therapeutic drugs in bodyfluids. The anti-asthmatic agent, theophylline, is a drug whosetherapeutic range is very narrow. An immunoassay for theophyllinerequires a highly specific antibody because of the occurrence in bodyfluids of other xanthines which are closely related structurally totheophylline and which, if recognized by the anti-theophylline antibody,would produce an erraneous value for the theophylline concentration inthe fluid being analyzed. Theophylline is 1,3-dimethylxanthine whilefour of the most commonly encountered cross-reactive xanthines are:caffeine, 1,3,7-trimethylxanthine; theobromine, 3,7-dimethylxanthine;xanthine; and hypoxanthine. The most frequently encountered xanthinewhich is a potential cross-reactant is caffeine which is ubiquitous inpopular beverages. It is also a metabolite of theophylline in neonatesin whom the level of caffeine may approach the level of theophylline. Itis thus essential that anti-theophylline antibodies employed in adiagnostic immunoassay for theophylline not cross-react with caffeine.

Theophylline and other compounds of formula weight generally less than1000 are not immunogenic unless coupled to a carrier which is itselfimmunogenic; see, for example, H. N. Eisen, Immunology, Harper and Row,1980. Such compounds are called haptens and there are numerous methodsknown in the art for coupling them to carriers in order to render thehapten immunogenic.

The choice of carrier and the site of attachment of the hapten to thecarrier are known to influence the immunogenicity of the hapten-carrierconjugate as well as the specificity of the antibodies produced. See,for example, B. F. Erlanger, Methods in Enzymology, Volume 70, 85(1980).

U.S. Pat. No. 4,156,081, issued May 22, 1979, to Singh, et al. describesthe synthesis of 3-substituted theophylline derivatives and their use asimmunogens to produce antibodies to theophylline which do notcross-react with caffeine. They do, however, cross-react with1-methylxanthine. Furthermore, there are large quantities of antiserumneeded for a commercial immunoassay and their production by animalimmunization is slow, laborious and not readily reproducible fromanimal-to-animal or even bleed-to-bleed in the same animal.

European Patent Application No. 44,441, published Jan. 27, 1982,discloses the production of monoclonal antibodies to drugs. It does notdisclose a monoclonal antibody to theophylline which substantially lackscross-reactivity with caffeine.

DISCLOSURE OF THE INVENTION

The cell lines of this invention are three continuous hybrid monoclonalcell lines, each capable of producing a unique monoclonal antibody totheophylline. The cell lines are hybrids of a spleen cell from a mouseimmunized with an 8-substituted theophylline-carrier conjugate(immunogen) and a mouse myeloma cell. Each of the antibodies of thisinvention exhibits less than 5% cross-reactivity with caffeine whenevaluated in a particle-enhanced turbidimetric inhibition immunoassayfor theophylline (described in copending patent application Ser. No.315,922, filed Oct. 28, 1981 and hereby incorporated by reference) andeach exhibits variable but low cross-reactivity with other xanthinesencountered in body fluids. The use of these monoclonal antibodies inimmunoassays for theophylline, especially in a particle-enhancedturbidimetric inhibition immunoassay, is also contemplated.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1, 2 and 3 are standard assay curves generated inparticle-enhanced turbidimetric inhibition immunoassays utilizingmonoclonal antibodies to theophylline produced by the cell lines 30/15,17/14, and 61/7, respectively.

DESCRIPTION OF THE INVENTION

Monoclonal antibodies are produced by fusing spleen cells from a mouse,immunized with the antigen or hapten of interest, in this casetheophylline, to a mouse myeloma cell line. When the compound to whichthe monoclonal antibodies are raised is a hapten, it is necessary tofirst conjugate the hapten to a high molecular weight carrier to obtainan immunogen. Such carriers include proteins, polysaccharides, andvarious latex particles. For the purposes of the present invention,theophylline is conjugated through its 8-position to amino groups onkeyhole limpet hemocyanin (KLH) using carbodiimide as a coupling agentto yield theophylline-8-KLH.

As a matter of practice, several immunizations can be performed atintervals over the course of several weeks to months. The immunizedanimal such as mouse is bled at the time of each injection and the serumassayed for presence of the desired antibody. Any suitable assay can beused; radioimmunoassays or enzyme-linked immunosorbent assays arefrequently employed. When antibody is detectable in the serum, theanimal is sacrificed and the spleen is removed aseptically for fusion.

Several different murine (mouse) myeloma cell lines are known to besuitable as fusion partners. The features of some of these cell linesare described in Current Topics in Microbiology and Immunology, referredto above. Generally, it is preferable to choose a myeloma line whichdoes not secrete an immunoglobulin product of its own.

Fusion is carried out most commonly by using polyethylene glycol as afusion promoter. Other fusion promoters such as Sendai virus can also beused. The ratio of spleen cells to myeloma cells can vary, most often aratio of 5-10:1 is used.

After fusion, the cells are diluted and cultured in a selective mediumsuch as hypoxanthine aminopterin thymidine (HAT). The unfused spleencells undergo a finite member of divisions and then die; and unfusedmyeloma cells will die, because the mutation which they carry in thehypoxanthine guanine phophoribosyl transferase (HGPRT) gene does notpermit them to survive on HAT medium. The suspension of hybrid andunfused cells is diluted prior to culturing so as to limit the number ofcells per vessel. Usually, they are diluted to the point at which thereare 1-5 cells per well of a microtiter plate.

When cell growth becomes visible, the culture supernatant is tested forthe presence of the desired antibody by solid-phase radioimmunoassay(RIA) or any other appropriate assay. The cells from wells producingantibody of interest are then cloned in soft agar or by limitingdilution to insure monoclonality.

Large volumes of antibody can then be obtained either by growing thehybridoma in vitro and harvesting the culture supernatant, or byinjecting the hybridoma cells into mice. Antibodies can be obtained inascites fluid by injecting the hybridoma cells into the peritonealcavity of pristane-primed syngeneic or congenic mice. Antibodies can beobtained in the serum by injecting the hybridoma intraveneously. Thequantity of antibody per milliliter is variable, usually being lowest inthe culture supernatant and highest in ascites fluid.

The monoclonal antibody thus generated can be characterized by itsimmunoglobulin class and subclass, as well as by its isoelectricfocusing pattern. Affinity constants can be obtained and the antibodycharacterized in terms of its cross-reactions with a panel of relatedantigens (haptens).

The present invention comprises monoclonal antibodies to theophylline.Three different clonotypes, 30/15, 17/14, and 61/7, were derived byimmunization of Balb/c mice with theophylline-8-KLH. All are products ofa single fusion. All are of the same heavy and light chain class buteach of these clonotypes has a unique isoelectric focusing pattern. Allbind theophylline and can be used in immunoassays for the measurement oftheophylline. One such immunoassay is a particle-enhanced turbidimetricinhibition immunoassay. None of these clonotypes cross-reactssignificantly with caffeine, the major interferent in immunoassays fortheophylline and each exhibits low cross-reactivity with otherxanthines.

The hybridoma cell lines 30/15, 17/14, and 61/7 were deposited in theAmerican Type Culture Collection, 12301 Parklawn Drive, Rockville, Md.20852 on Aug. 5, 1982 and were given the ATCC accession numbers HB8152,HB8153, and HB8154, respectively.

The hybridoma cell lines producing the monoclonal antibodies of thisinvention are hybrids of the mouse myeloma line P3-NSI-1-Ag4-1 (referredto as NS-1) and Balb/c spleen cells. The NS-1 cell line is itselfderived from a Balb/c mouse and synthesizes a kappa light chain which,however, is not secreted. NS-1 is available from the Salk Institute CellDistribution Center, La Jolla, Calif.

The hybridoma cell lines of this invention are hybrids, a fact evidencedby the presence of gene products of both parents in each hybrid clone.These are characterized by the specific antibody which each produces.Each cell line has been shown to behave in a stable fashion for at leastone year with respect to the specificity of the antibody produced andother assay performance parameters. The 30/15 line has been re-clonedfrom frozen material on two occasions; the 17/14 line has been re-clonedfrom frozen material once. The 61/7 line has not yet been re-cloned.

EXAMPLE 1 Production of Monoclonal Anti-Theophylline Cell Lines A.Preparation of Theophylline-8-KLH

8-(3-carboxypropyl)-theophylline was synthesized as described incopending patent application Ser. No. 315,922, filed Oct. 28, 1981. 15mg of this compound was reacted with 7 mg of N-hydroxysuccinimide in 2.0ml of dimethylformamide at 4° C. for 18 hours. At the end of this time,200 mg of KLH, dissolved in 15 ml of 0.1M sodium carbonate, pH 8.5, wasadded. The reaction mixture was stirred for 18 hours at 4° C. Unreactedtheophylline was removed by dialysis.

B. Immunization

A Balb/c mouse was injected intraperitoneally with 300 μg oftheophylline-8-KLH (prepared as in (A) above) emulsified in 0.3 mL ofcomplete Freund's adjuvant. Three booster injections were given at21-day intervals as described above. Seven days after the last boost,the mouse was bled and the serum tested for circulatinganti-theophylline antibody by solid phase RIA using I¹²⁵ -labeledprotein A and by a particle-enhanced turbidimetric inhibitionimmunoassay, described below. The mouse was given a final boostintraperitoneally and four days later the spleen was removed for fusion.

C. Particle-enhanced Turbidimetric Inhibition Immunoassay

This assay was used to screen the mouse serum, as follows: 5 μL of mouseserum was added to a cuvette containing 12.5 μL of theophylline-HSAcoated latex particles (described in copending patent application Ser.No. 315,922, filed Oct. 28, 1981), 2.5% (v/v, final concentration)polyethylene glycol 6000, and 0.15M phosphate buffer, pH 7.8, in a totalvolume of 1 mL. Turbidity due to antibody-mediated aggregation of thelatex particles was measured at 340 nm, 37° C., in a recordingspectrophotometer. The rate of turbidity formation was 0.2 absorbanceunits for 5 μL of serum from the mouse whose spleen was subsequentlychosen for fusion.

D. Fusion

The spleen was removed aseptically and a single cell suspensionprepared. The cells were then fused with NS-1 myeloma cells at a ratioof 5:1 using 0.2 mL of 30% (v/v) polyethylene glycol 1000 in serum-freemedium. The fused cells were washed in serum-free medium, suspended in30 mL of serum-free medium, plated into 96-well microtiter plates (about50 μL per well) containing feeder layers of mouse peritonealmacrophages.

HAT selection medium was added 18 hours later. After five days, thewells were scanned every other day for the presence of hybrid cellcolonies. When hybrids were detected (approximately 2 weeks afterfusion), the wells were marked and kept under observation until thecells had grown to the point at which expansion into larger volume wasdesirable. At this time, the supernatants from the hybrid-containingwells were harvested for screening and the cells were expanded into24-well plates. After 3 weeks, the hybrids were cultured on HT medium(containing hypoxanthine and thymidine). At this stage and macrophasefeeder layers had cleared the cultures of dead cells and debris andtheir use was discontinued.

E. Screening

The supernatants harvested above were screened for anti-theophyllineantibody by solid state RIA. The antigen used in the screening assay wastheophylline-8-BSA (20 moles of theophylline per mole of BSA),synthesized as described above for theophylline-8-KLH. The secondantibody was I¹²⁵ -labeled goat anti-mouse Ig.

A total of 56 positive wells was detected by RIA. These wells were thenrescreened using I¹²⁵ -labeled protein A. This reagent detects onlyantibodies of the IgG class. Of the 56 positive supernatants, 28 provedto be of the IgG class, including cell lines 30/15, 17/14, and 61/7.

These 28 supernatants were then screened for their ability to bind freetheophylline. Free theophylline (final concentration 10 μg/mL) wasincorporated into the solid state RIA; the ability to bind freetheophylline was evidenced by a decrease in antibody binding to theimmobilized theophylline-8-BSA antigen.

Supernatants were screened in a similar manner for their ability to bindcaffeine. In these assays, free caffeine was present at a finalconcentration of 50 μg/mL. Lack of cross-reactivity with caffeine wasevidenced by uninhibited binding of the antibody of this invention tothe immobilized theophylline-8-BSA antigen.

Table I shows the results of these assays for the antibodies produced bythe three cell lines of interest. The data show that the binding ofthese antibodies to immobilized theophylline-8-BSA is inhibited by freetheophylline but not by 5-times as much free caffeine.

                  TABLE I                                                         ______________________________________                                        Antibody Specificity                                                                       CPM Bound I.sup.125 Labeled                                                   Protein A                                                        Monoclonal antibody                                                                          30/15       17/14   61/7                                       ______________________________________                                        Control (no free                                                                             1245        1195    1443                                       theophylline)                                                                 Theophylline added                                                                            800         687    1134                                       Caffeine added 1316        1265    1483                                       ______________________________________                                    

F. Cloning at Semi-Limiting Dilution

The hybrid cell lines of interest (30/15, 17/14, 61/7) were expanded tosufficient numbers to allow cloning at semi-limiting dilution, that is,at approximately 3 cells/microtiter well. An aliquot of the cells wasalso viably frozen in liquid nitrogen as a safeguard against loss.Feeder layers of peritoneal macrophages were again used. When acceptablenumbers of cells were present in the wells, the supernatants were againscreened using the solid phase RIA. Positive wells were selected forexpansion and further cloning.

G. Cloning at Limiting Dilution

The wells selected were then cloned at limiting dilution, using strictPoisson statistics. In this case approximately one-third of the wellsshould show growth and the probability is very high that the cellsgrowing in a given well were the progeny of a single hybridoma cell.When sufficient numbers of cells were present in the wells, thesupernatants were again tested for presence of monoclonal antibody. Alllines continued to produce the desired antibody.

H. Chain Composition

All monoclonal antibodies derived from cell lines 30/15, 17/14, and 61/7consisted of gamma heavy chains (subclass 1) and kappa light chains, asdetermined by double diffusion in agar gel.

EXAMPLE 2 Production of Monoclonal Antibody in Ascites Tumors

In order to produce large amounts of monoclonal anti-theophylline,approximately 10⁶ hybridoma cells were injected intraperitoneally intopristane-primed syngeneic Balb/c mice. Ascites fluid was withdrawn andshown to have high concentrations of anti-theophylline activity by bothsolid state RIA and a particle-enhanced turbidimetric inhibitionimmunoassay, as described below.

EXAMPLE 3 Immunoassay for Theophylline

Ascites fluid from 30/15, 17/14, and 61/7-primed mice was used in aparticle-enhanced turbidimetric inhibition immunoassay performed on theaca™ discrete clinical analyzer (available from E. I. du Pont de Nemoursand Company). 20 μL of a serum-based theophylline calibrator, containingfrom 0 to 40 μg/mL of theophylline, was automatically injected into ananalytical test pack (described in No. Re 29,725, issued Aug. 8, 1978 toJohnson et al., hereby incorporated by reference) in the filling stationof the instrument, followed by 4.980 mL of buffer containing 3% (v/v)polyethylene glycol 6000, 0.1% GAFAC RE-610, and 0.15M phosphate, pH7.8. The pack was automatically warmed to 37° C. 40 μL oftheophylline-HSA coated latex particles (described in copending patentapplication Ser. No. 315,922, filed Oct. 28, 1981) and 50 μL of 750 mMdithioerythritol were added from dimples 2 and 3, respectively, atbreaker/mixer I. Anti-theophylline antibody, as sterile-filtered ascitesfluid, was added to the pack from dimple 6 at breaker/mixer II (17-50μL, depending on the cell line). The rate of turbidity formation wasmeasured automatically at 340 nm, approximately 39 seconds after theinitiation of the reaction at breaker/mixer II.

FIG. I shows a typical standard curve generated using 28 μL of 30/15ascites fluid. FIG. 2 shows a typical standard curve generated using 17μL of 17/14 ascites fluid. FIG. 3 shows a typical standard curvegenerated using 50 μL of 61/7 ascites fluid.

EXAMPLE 4 Specificity of Cell Lines 30/15, 17/14, and 61/7 forTheophylline

In order to measure theophylline accurately in an immunoassay, theanti-theophylline antibody must exhibit little or no cross-reactivitywith other xanthines present in samples. Cross-reactivity is defined asthe percentage error in measurement introduced when the material inquestion is present at a final concentration of 10 μg/mL in a samplecontaining 10 μg/mL theophylline.

Table II compares the cross-reactivities of 30/15, 17/14, 61/7 with tworabbit antisera, 924P0213 and 904P0518, to caffeine, theobromine,1,7-dimethylxanthine, 3-methylxanthine, hypoxanthine, and xanthine. Thetwo rabbit antisera were raised to theophylline-8-BSA and are typical ofmore than ten different pools of rabbit antisera tested.

The monoclonal antibodies of this invention produced by cell lines30/15, 17/14, and 61/7 are specific for theophylline and are superior tothe polyclonal rabbit antibodies.

                  TABLE II                                                        ______________________________________                                                 Cross-Reactivity (% Error)                                                    Monoclonal    Rabbit                                                 Compound   30/15   17/14   61/7  924P0213                                                                             904P0518                              ______________________________________                                        Caffeine   5       5       5     40     25                                    Theobromine                                                                              5       30      25    --     --                                    1,7-Dimethyl-                                                                            10      25      10    --     --                                    xanthine                                                                      3-Methylxanthine                                                                         5       5       5     --     --                                    Hypoxanthine                                                                             5       5       10    --     --                                    Xanthine   5       5       5     --     --                                    ______________________________________                                    

I claim:
 1. Mouse monoclonal IgG isotype antibody to theophylline having5% or less cross-reactivity with caffeine wherein the hybrid continuouscell line producing said antibody is a hybrid of a spleen cell from amouse immunized with an 8-substituted theophylline-carrier conjugate anda mouse myeloma cell line.
 2. The monoclonal antibody of claim 1 having30% or less cross-reactivity with theobromine and 5% or lesscross-reactivity with 3-methylxanthine.
 3. The monoclonal antibody ofclaim 1 wherein the conjugate is theophylline-8-KLH.